Lateral flow assay devices and methods are known in the art. Previously, such devices have been developed to test samples that are easily available in large quantities. However, when the test sample is blood or a component of blood, the collection of a large sample is not always possible, particularly at a point of care such as a doctor's surgery.
Generally these devices comprise a lateral flow matrix, for example, nitrocellulose membranes and the like. A sample applied to the matrix flows along the matrix, and one or more analytes within the sample react with one or more reagents within the lateral flow matrix. Typically, at least one of these reagents is immobilized within the matrix allowing any reaction with the analytes to be detected, for example, visually. Unfortunately, variations associated with sample transfer and diffusion of the sample to the membrane result in a flow that is largely uncontrolled and uneven before reaching a test area. This is because such devices rely on capillary action of the fluids alone. Such a reliance on capillary action may have an adverse affect on the accuracy of the device because the amount of analyte and/or label captured across a test area is not consistent. The use of capillary action alone also means that assays are slow because of unreliable fluid wicking. The assays are also unsuitable for small fluid samples, such as in nucleic acid detection, where the membrane may dry before the assay is completed or there may be insufficient fluid to travel the length of the test device.
As such, a need currently exists for a simple and efficient technique for improving lateral flow assays, particularly to allow faster testing whilst enabling low volume tests to be performed.
One area where lateral flow point of care devices would be useful is in the field of cholesterol and blood lipid testing.
It is well known that the concentration of various lipoproteins in the blood is correlated with the risk of an individual developing atherosclerosis. Atherosclerosis is a disease that affects arterial blood vessels and is commonly referred to as a “hardening” or “furring” of the arteries. It is caused by the formation of multiple plaques on the blood vessel walls, in large part due to the accumulation of macrophage white blood cells and promoted by low density lipoproteins. Without adequate removal of fats and cholesterol from the macrophages by high density lipoproteins (HDL), a chronic inflammatory response develops in the walls of the arteries.
Most of the circulating cholesterol in blood plasma is found in three major classes of lipoproteins. Cholesterol and cholesterol esters are water insoluble substances and are therefore carried by these lipoproteins within the circulatory system for eventual utilisation by the cells of the body.
Each of these lipoprotein classes carries varying amounts of cholesterol. Total serum cholesterol is therefore a complex average of the amount that each lipoprotein class contributes to the total lipoprotein concentration of the serum.
Each class of lipoprotein plays a different role in atherosclerosis. High density lipoproteins or HDL are generally regarded as being ‘good cholesterol’, that is they are anti-atherogenic. In contrast, Low density lipoproteins or LDL are often referred to as ‘bad cholesterol’ since they are known to be highly atherogenic. Another class of lipoproteins, very low density lipoproteins or VLDL are considered to be slightly atherogenic.
Levels of HDL in the blood have been extensively investigated in view of the inverse relationship between HDL cholesterol and the risk of atherosclerosis and, for example, heart attack. Thus, if the levels of HDL cholesterol are determined to be low, an individual may have an increased risk of developing atherosclerosis. Therefore, this risk can be estimated by assaying HDL cholesterol. From these assay results an approximate amount of LDL cholesterol may be calculated using the following equation:LDL Cholesterol=Total Cholesterol−⅕ Total Cholesterol−HDL Cholesterol
To determine the cholesterol content of the various cholesterol fractions, generally four methods have been used. These include (1) ultracentrifugation, (2) fractional precipitation, (3) calculation using the Friedewald equation and (4) electrophoretic separation and precipitation.
Each of these methods suffers from a number of drawbacks. For example, ultracentrifugation requires the use of specialised laboratory equipment and may take several days to complete. Fractional precipitation and electrophoretic separation are both time-consuming and again require the use of specialised equipment. The Friedewald equation is inaccurate because it estimates the concentration of LDL cholesterol by subtracting the cholesterol associated with other classes of lipoproteins. Thus, the equation provides an indirect estimation based on three independent lipid analyses each of which provides a potential source of error.
As a result of these drawbacks, the results of cholesterol assays are not available for several hours or even days and cannot be performed in smaller laboratories or by doctors in their surgeries. Accordingly there is a need for a device and method of performing a cholesterol assay that is both simple and inexpensive to use. There is also a need for an assay that directly measures the concentration of each class of lipoprotein without relying on indirect estimates.
When solid hydrophobic molecules are added to an aqueous solution or suspension they form an immediate precipitate and do not enter the aqueous phase because the hydrophobic molecules ‘stick’ together rather than dissolve. Even with vigorous stirring of the precipitate the number of direct encounters between the hydrophobic molecules and molecules in solution is small. Such interactions may also be thermodynamically unfavourable.
Methods of the prior art involve dissolving hydrophobic molecules in suitable water-miscible organic solvents prior to mixing with an aqueous solution or suspension. On entering the aqueous solution, the hydrophobic molecules are mono-dispersed and therefore have an increased probability of interacting with the molecules already in solution. However, such methods suffer from the drawback that the organic solvent is often toxic or can interfere with enzymatic reactions or fluorescence measurement. Such methods are generally also not suitable for point of care use such as in a Doctor's surgery or clinic.